**4. Biohydrogen and methane production in two-stage fermentation process**

Hydrogen is an eco-friendly, clean energy alternative because its combustion by-product is only water, so does not contribute to the greenhouse effect. Hydrogen has a high energy yield (122 kJ g-1), therefore in recent times a great deal of attention is being paid to the usage

and denaturate enzymes (Liu et al., 2007; Pitt & Ross, 2003). Low energy ultrasounds can produce a variety of effects on biological materials, including the inhibition or stimulation cellular metabolisms, enzyme activity, alteration of cell membranes and other cellular structures (Liu et al., 2007; Liu et al. 2003a). According to Xie et al. (2008), cavitation is the primary basis of biological effects of low intensity ultrasound. Cavitation bubbles produced by low intensity ultrasound can cause acoustic microstreaming (Xie et al., 2008). The microstreaming surrounding the cells can cause shear stress and enhance the mass transfer, which may stimulate metabolic activities inside the cells (Liu et al., 2003b; Pitt & Ross, 2003; Xie et al., 2008). When ultrasonic intensity is sufficiently low, a stable cavitation occurs and leads to the enhancement of mass transfer and fluid mixing, which produces positive effects

The growth activity of yeast cells is hardly changed within the early period of sonication regardless of either damage to cell wall, or complete inactivation of the yeast located in the cavitation zone (Tsukamoto et al., 2004). Short sonication time up to 5 min of irradiation indicated bactericidal effects, but the cells were able to repair the damages. According to Guerrero et al. (2005) yeasts, inclusive with *S. cerevisiae*, are highly resistant to ultrasound damage. Moreover, at relatively low intensity of ultrasounds, microorganisms can adapt to the irradiation exposure and their biological activity increases (Liu et al., 2007). With relatively short irradiation period, cell damage and membrane permeability induced by ultrasounds appear to be temporary and reversible. Lanchun et al. (2003) also stated that

The utilization of milk permeate to ethanol in continuous fermentation by co-immobilized *S. cerevisiae* is possible. The optimal ultrasonic intensity and irradiation period are varied in each biological process enhanced by ultrasound and should be find experimentally. According to this experiment, stimulation of yeasts activity could be achieved in the presence of low intensity ultrasound (1 W L-1, 20 kHz), and 1 min every 6 h irradiation period is favorable to increase ethanol production efficiency. Moreover, the short exposure of yeast to ultrasound could reduce the operation costs comparing with continuous

For the continuously operating bioreactors, the maximum rates of sugar utilization were 98.9 and 92.4% for the yeast with ultrasound exposure and without ultrasound exposure (p<0.05), respectively*.* The maximum ethanol yield was 0.532 g g-1 lactose, while using *S. cerevisiae* without ultrasound exposure 0.511 g g-1. The study showed that there is no need to extend the HRT over 36 h or more, because most of the lactose was converted into ethanol

All results obtained here raises the new perspectives for disposal UF whey permeate.

**4. Biohydrogen and methane production in two-stage fermentation process**  Hydrogen is an eco-friendly, clean energy alternative because its combustion by-product is only water, so does not contribute to the greenhouse effect. Hydrogen has a high energy yield (122 kJ g-1), therefore in recent times a great deal of attention is being paid to the usage

during 24 h (95.6% in the ultrasound-assisted fermentation).

on the rate of biological reactions in the exposure systems (Liu et al., 2007).

sonication cannot influence on fermentation strength of *S. cerevisiae* descendants.

**3.2.3 Conclusions** 

irradiation.

of hydrogen as a fuel. However, a major doubt on hydrogen as a clean energy alternative is that most of the hydrogen gas is currently generated from fossil fuels by thermochemical processes, such as hydrocarbon reforming, coal gasification and partial oxidation of heavier hydrocarbons (Castellό et al., 2009; Mohan et al., 2007). These methods are considered to be energy intensive and not environmental friendly. It is well known that only biological hydrogen production processes from the fermentation of renewable substrates, such as organic wastewater or other wastes are the promising alternative for hydrogen generation. Several strategies for the production of biohydrogen by fermentation in lab-scale have been found in the literature: photo-fermentation (Gadhamshetty et al., 2008)**,** dark-fermentation (Krupp & Widmann, 2009) and combined-fermentation, which refers to the two fermentations combined (Nath & Das, 2009). However, no strategies for industrial scale productions have been found. In order to define the industrial scale biohydrogen production, more information from laboratory scale experiments are needed, especially related to design and optimization process, and operating parameters. Moreover, generation of biohydrogen by acidogenic phase of anaerobic process (dark-fermentation) is connected with incomplete degradation of organic material into organic acids, so there is a need to utilize by-products of the fermentation process.

As a result, the fermentative hydrogen production could be coupled with subsequent anaerobic digestion step with the conversion of remaining organic content to biogas. A twostage fermentation process, in which acidogenesis and methanogenesis occur in the separate reactors may offer several advantages, such as improved total wastewater degradation and enhancing biohydrogen and methane production (Venetsaneas et al., 2009).

The dairy industry produces highly concentrated, carbohydrate-rich wastewaters, but their potential for biohydrogen generation has not been extensively studied. There were some experiences working with cheese whey as the substrate for biohydrogen production (Azbar et al. 2009; Castellό et al., 2009; Venetsaneas et al., 2009). The objectives of this work were: (1) to check the ability to produce biohydrogen using raw, unsterilized UF whey permeate, (2) to combine biohydrogen dark-fermentation process with methane fermentation of biohydrogen production by-products (mainly organic acids) in two-stage continuous fermentation process.
